Process for production of diketopiperazine dihydroxamates and intermediates therefor

ABSTRACT

A process for the production of diketopiperazine dihydroxamates which are useful in the biological field and intermediates for the synthesis of said diketopiperazine dihydroxamates is disclosed.

United States Patent lsowa et al.

1451 Aug. 26, 1975 PROCESS FOR PRODUCTION OF DIKETOPIPERAZINE DIHYDROXAMATES AND lNTERMEDlATES THEREFOR Inventors: Yoshikazu lsowa, Tokyo; Toshiyuki Takashima, Ebina; Muneki Ohmori, Sagamihara; Hideaki Kurita, Sagamihara; Masanari Sato, Sagamihara; Kaoru Mori, Sagamihara, all of Japan Assignee: Sagami Chemical Research Center,

Tokyo, Japan Filed: Aug. 2, 1973 App]. No.: 384,847

Related US. Application Data Division of Ser. No. 239,349, March 29, 1972, Pat. No. 3,772,265,

Foreign Application Priority Data Mari 30, 1971 Japan 4648373 Primary Examiner-Donald G. Daus Assistanl Examiner-Jose Tovar Attorney, Agent, or FirmSughrue, Rothwell, Mion, Zinn and Macpeak 57 ABSTRACT A process for the production of diketopiperazine dihydroxamates which are useful in the biological field and intermediates for the synthesis of said diketopiperazine dihydroxamates is disclosed.

7 Claims, No Drawings PROCESS FOR PRODUCTION OF DIKETOPIPERAZINE DIHYDRUXAMATES AND INTERMEDIATES THEREFOR COR I N/ RCO\ N Ho /\N wherein R represents a lower alkyl group having l to 4 carbon atoms. inclusive. The synthesis of compounds of this type has not been reported in literature. and it was confirmed that this type of compound exists only in the natural field. For e\ample. rhodotorulic acid which is included in the product of this invention (R methyll was reported by C. L. Atkin, et al. Bio

ill

chemistry. 7. No. It]. 3734 [968) in which rhodoto rulic acid was isolated from the fermentation broth 01 a red yeast identified as R/rmlumrulu pililmmuu and the structural formula of rhotlotorulic acid was determined as cyclo-di-N 5 -acetyl N -h vdroxy-L ornithyl. it was also reported in Biochcmistry'fl 7. No. l(), 3734 1968) that rhodotorulic acid exhibits a biological activity comparable to that of schizokinen in Lankfords Bacillus system and that it also exhihits an activity as a growth factor in the assay using Arthrohacter species. Thus. rhodotorulic acid is an extremely useful compound in the biological field and the success in synthesis of rhodotorulic acid is of significant importance in industry.

This invention also contemplates certain novel pep tides which are useful not only as intermediates for the synthesis of cyclo-di-N acetyl-N -hydroxy ornithyL but also are biologically active. Peptides isolated from the metabolites of microorganisms fre quently contain. as their components urhydroxyamino-a-aminoacids and therefore the novel amino acid derivatives represented by the formula lll below are expected to be useful compounds for various purposes such as the synthesis of a wide variety of biologically active compounds.

The object of this invention is therefore to provide a process for the production of diketopiperazine hydroxamate derivatives.

Another object of this invention is to provide novel intermediates for the synthesis of diketopiperazine hy droxamate derivatives and a process for the production of such intermediates.

The process according to the present invention can be illustrated by the following reaction scheme:

From (ll RCO herein R represents at protecting group as defined :reinafter. and R is a lower alkyl group having I to 4 irhon atoms inclusive.

The starting materials of the process of this invenm. N tosyl-N -benzyloxy-l.-ornithine having the 'rmula I and N -tosyl-N -benzyloxy-L-ornithine kyl ester having the formula II. can be derived from -(N-tosyl-N-benzyloxy)-ornithine which is in turn, )ltllfltlhlti by hydrolysis of the a-acylamino-cu-(O N- substituted-hydroxyJ-alkylmalonic acid ester disosed in Japanese Pat. No. 24763/1970 proposed by re present inventors. More specifically the starting aterial having the formula I can be produced by first lbjecting the above N -tosyl-N -ben7.yloxy-DL- 'nithine to optical resolution to obtain the correionding L-amino acid and then protecting the -amino group of the L-amino acid with a protecting 'oup. The term protecting group used herein means COR (VIII) any one of the conventional protecting groups for the amino group commonly employed in the art of peptide synthesis and includes. for example. carbobenzoyl group. a t-butoxycarbonyl group. a p-methoxybenzyloxycarbonyl group and the like Another starting material having the formula ll (a lower alkyl ester) can easily be produced from the above L-amino acid by the conventional ester formation technique, for example, by suspending the L-amino acid in an appropriate alcohol and passing hydrogen chloride gas into the resulting alcoholic suspension.

The reaction between the starting materials (I and Il) can be accomplished by coupling the materials in an approximately molar equivalent in accordance with the acid anhydride method or the isoxazoline method in a solvent such as tetrahydrofuran. nitromethane or the like, at a temperature in the range of from 5"C to l()C thereby yielding an L-hydroxyornithine dipeptide derivative represented by the formula III. The subsequent removal of the protecting group from the resulting compound (III) can be carried out by any one of the well-known procedures. such as by treating the compound (III) while stirring in an acetic acid solvent in the presence of a hydrogen halide. The acetic acid solvent may preferably be acetic acid per se or an acetate ester. The removal of the protecting group can be conducted at room temperature. Le, a temperature between about C and C. Under such conditions, the desired dipeptide ester having the formula IV can be obtained in the form of its hydrohalide salt in high purity and high yield. It is to be understood that the above coupling reaction and the removal of protecting group may also be adapted to the corresponding D-form thereby resulting in the production of the desired compound (IV) in the D-form The cyclization of the thus obtained dipeptide ester of the formula IV can then be conducted in an appropriate alcohol using ammonia gas to produce cyclodi- N -tosyl-N -ben7.yloxy-ornithyl of the formula V. For example, the cyclication may be carried out by adding the dipeptide ester in the form of its hydrohalide salt to methanol which has previously been saturated with anhydrous ammonia and allowing the mixture to stand for several days at a temperature of from about (1C to room temperature.

The resulting compound (V) is then subjected to detosylation procedure to give a novel compound having the formula (VI), cyclo-di-N -ben7.yloxyornithyl. The detosylation can be effected by treatment of the compound of the formula V in a specific solvent system. The solvent system used in the detosylation step comprises hydrogen bromide, acetic acid and phenol in such a proportion that hydrogen bromide in acetic acid is a solution containing hydrogen bromide in an amount of to by weight. preferably about 367: by weight and that phenol is used in an amount of from 0.1 to 0.3 parts by weight per l part by weight of acetic acid. The detosylation can be accomplished by adding the compound (V) to the above hydrogen bromide-acetic acidphenol solvent system and stirring the mixture at approximately room temperature, generally in the range of from |8C to 25C, for at least 30 hours, generally from 30 to hours. At lower temperatures the reaction does not proceed smoothly, while at higher temperatures the desired detosylated compound (VI) tends to decompose. Unduly prolonged stirring also results in the decomposition of the desired compound (VI). After completion of the reaction, the compound (VI) can be isolated as crystals from the reaction mix ture by any one ofthe well-known techniques. It is also to be understood that the detosylation procedure may be adapted to the compound (V) in either D-, L- or DL-form thereby yielding the corresponding optical isomer of cyclo-di-N -ben2yloxyornithyl having the formula (VI).

The thus obtained compound (VI) can then be acylated with an acid anhydride of lower aliphatic acids in a basic solvent to give cyclo-dhN acetyl-N ben7yloxy ornithyl having the formula VII. The acetylation can easily be conducted by any one of the wellknown acylation procedures, and the solvent of choice for the acylation includes quinoline and py ridinc. Again, the acylation may be employed for the conr pound (VI) in either D-, L- or DL-form thereby yielding the corresponding optical isomer of the acylate (VII).

The final step of the process of this invention is debenzylation of the above acylate (VII). This debenzylation can be carried out by catalytic reduction in the presence of a catalyst, for example, palladium-oncarbon, and the like, at normal temperature under normal pressure. The amount of the catalyst is not critical and may be a so-called catalytic amount. The debenzylation is usually completed within about 10 to 20 hours at normal temperature. The desired final product of the formula VIII, cyclo-di-N -acetyl-N 5 -hydroxyornithyl, can be isolated from the reaction mixture in a usual manner and purified by recrystallization, etc. The debenzylation is also employed for the compound (VII) in either D-, L- or DL-form thereby yielding the corresponding optical isomer of the product having the formula VIII.

The present invention is further illustrated by the following examples, but they are not to be construed as limiting the scope of the invention.

Example I (A). N-Benzyloxycarbonyl-N -tosyl-N -benzyloxy- L-ornithyl-N -tosyI-l\l -benzyloxy-L-ornithine methyl ester L26 ml of isobutyl chloroformate was added to a cooled solution of L3] ml of triethylamine and 4.90 g of N" -benzyloxycarbonyl-N -tosyl-N benzyloxy-bornithine in 45 ml of tetrahydrofuran at a temperature of from l() to -l5C. After 20 minutes, to the resulting solution was added a solution of 4.24 g of N -tosyl-N' -benzyloxy-L-ornithine methyl ester hydrochloride and L3] ml of triethylamine. The mixture was then stirred overnight at room temperature, and evaporated in vacuo. The residue was combined with water, and the mixture was filtered to collect the product. The thus obtained product was dissolved in 50 ml of ethyl acetate, and the solution was washed successively with lN hydrochloric acid. water, 0.5M aqueous sodium bicarbonate and water and dried over anhydrous sodium sulfate. The solution was then filtered and evaporated in vacuo to dryness to give a product identified as N" -benzyloxycarbonyl-N tosyl-N benzyloxy-L-ornithyl-N -tosyl-N -benzyloxy-bornithine methyl ester by its N.M.R. and IR. spectra.

Yield: 8.4 g (9871).

Analysis: Calcd. for C H N O S C, 61.70; H, 5.90; N, 6.13

Found: C, 6L5); H, 6.12; N, 6.25.

(B). N tosylhl' -benzyl0xy-L-ornithyl-N -tosylbenzyloxy-L-omithine methyl ester hydrobromide 8.2 ml of 36% hydrogen bromide-acetic acid was added to a solution of 8.3 g of N -benzyloxycarbonyl- N -ben7.yloxy-L-ornithine methyl ester obtained in Example I, (A) in 6.5 ml of acetic acid. After allowing to stand for minutes, the resulting solution was cvaporated in vacuo. and the residual oily substance was triturated with ether and repeatedly washed with ether by decantation. The resulting oily substance was identitied as N -tosyl-N -henZyloxyL-ornithyl-N 4 tosyl-N -benzyloxy-l.-ornithine methyl ester hydrobromide by its thin layer chromatography.

Yield: 6.3 g (79)? Example 2 A. A). N" -t-Butylxycarbonyl-N -t0syl-N' -benzyloxy- L-ornithyLN tosyl-N -benzyloxy-Lornithine methyl ester 3.82 ml of triethylamine was added to a suspension If 12.26gofN -t-butyloxycarbonyl-N -tosyl-N ienzyloxy-L-ornithine and 6.3l g of N-cthyl-5- ihenylisoxazolium-3'-sulfonate in 80 ml of nitrometh- .ne charged into a sealed glass vessel at a temperature If 0C. The mixture was stirred in an ice bath until a :lear solution was obtained. ie. for two hours. To the esulting solution was then added 1 L g ofN -tosyl- \l 5 -benzyloxy-L-ornithine methyl ester hydrochloide and 3.82 ml of triethylamine at a temperature of )C. The reaction mixture was stirred for two hours at )C and then overnight at room temperature, and eviorated in vacuo to give an oily substance. The thus obained residue was extracted with ethyl acetate. and the :thyl acetate extract was washed with water. an aque- )LlS sodium carbonate (three times) and finally water 'ollowcd by drying over magnesium sulfate. After filtraion. the filtrate was evaporated in vacuo. and the oily 'esidue was triturated with petroleum ether. The thus ibtained product was purified by dissolving in ethyl ac- :tate and adding petroleum ether to give a purified roduct identified as N tbutyloxycarhonyl-N osyl-N -henzyloxy-L-ornithyl-N -tosyl-l l -ben- :yloxy-L-ornithine methyl ester by its N.M.R. and LR. .pectra. Melting point. 6369C'; [a],, "+l.l (C. l in :thyl acetate).

Analysis: Calcd. for C H N.O S

C. 59.98; H. 6.41; N. 6.37

Found: C. 60.09; H. 6.65; N. 610.

:8). N -tosyl-N -benzyloxy L-ornithyl-N' t0syl- N -benzyloxy-L-ornithine methyl ester hydrochloride 80 ml of 3N hydrochloric acid in ethyl acetate was idded to a solution of 18.76 g of N -twutyloxycarbonyl-N -tosyl-N -benzyloxy-L .irnithyl-N -tosyl-N -hen7.yloxy L-ornithine methyl :ster obtained in Example 2 (A). The resulting solution was allowed to stand at room temperature for tvvo tours and evaporated in vacuo to give an oily substance .vhich was then triturated with either to crystallize. The .hLlS obtained crystalline product was identified as N tosyl-N" -benzyloxy-L-ornithyl-N -tosyl*l\l venzyloxy-L-ornithine methyl ester hydrochloride by ts N.M.R. and l.R. spectra.

Yield: 16.10 g (92.5)? Melting point. l00-l05C. :al +l2.l() (c. l in methanol).

Analysis: Calcd. for C;..,H,.,N,O,,S Cl:

C. 57.30; H. 6.04; N. 6.85

Found: C. 56.98; H. 6.24; N. 6.58.

Example 3 (A). N t-ButyloxycarbonyLN -tosyl-N -benzyl0xy- D-ornithyl-N -tosyl-l l benzyloxy-D-ornithine methyl ester The reaction described in Example 2 (A) was carried Jut under the same conditions but using N" -t- Ill (it l butylozt. carbonyl-N -tosyl-N -benzyloxy-D-0rnithine and N -tosyl-l l -benzyloxy-D-ornithine methyl ester hydrochloride in place of N -t-butyloxycarbonyl-N -tosyl-N -benzyloxy-L-ornithine and N' -t0syl-N -benzyloxy-L-0rnithine methyl ester hydrochloride, respectively, to obtain N -tbutyloxy-carbonyl-N -tosyl-N -benzyl0xy-D-ornithyl8LN tosyl-l-l" -benzyl0xy-D-ornithine methyl having a melting point of 6368C.

Yield: 90.3%. [ot],, l.4 (c. l in ethyl acetate). Analysis: Calcd. for C, ,H -...N.O S

C. 59.98; H, 6.41; N. 6.37

Foundi C. 60.01; H. 6.69; N. 6.03.

(B). N -t0syl-l' l" -benzyloxy-D-ornithyl-N -tosyl- N -ben7.yloxy-D-ornithine methyl ester hydrochloride Example 4 3.95 g of N -tosylN benzyloxy-l.ornithyl- N -tosyl-l l -ben2yloxy-l.ornithine methyl ester hydrochloride prepared as in Example 1 (B) was added to ml of methanol previously saturated with anhydrous ammonia at a temperature of 0C. and the mix- Cyclo-di-N tosyl-N -benzyloxy-L-ornithyl ture was allowed to stand in a glass vessel for two days at room temperature. The crystals l.l g) formed during the standing were filtered. and the filtrate was concentrated to dryness. The resulting crystalline residue was combined with water followed by filtration. and recrystallized from methanol to give cyclo-di-N -tosyl N -benzyloxy L-ornithyl.

Yield: 54%.

Melting point. l76l77C. dimethyl-iormamide Analysis: Calcd. for C;.,.H,.N,O,.Sv

C. 60.94; H. 5.92; N. 7.48; S. 8.56

Found: C. 6l.l5; H. 5.85: N. 7.48; S. 8.59.

(B). Cyclo-di-N -benzyloxyL-ornithyl Dihydrobromide 1.88 g of cyclo-di-N' -tos vll\l 5 -benzy|oxy-l.- ornithyl prepared as in Example 4 (A) was added to a solution of 5 g of phenol in 20 ml of 36% hydrogen bromide-acetic acid at room temperature. After stirring for 50 hours at room temperature. the mixture was evaporated in vacuo at a temperature of 30C. and the residual oil substance was crystallized by adding cthcr. The resulting crystals were filtered and washed successively with ethanol and ether to give the desired product. A portion of the product was recrystallized from cthanol for analysis to give cyclo-di-N -ben7.yloxy-l.

orinth l liydrolrromide having a melting point of lt 3-llt'1"(j.

Analysisv Calcd. for Hn- NfihlHBr:

C. 48.111; H. 5.37; N. 9.33

Found: (1 473)]: H. 5.81; N. 9. Ill.

The product was identified. by its l\l.M.R. and IR. spectra. as a compound having the following structural formula;

7ation of the residual crystals from boiling water yield ing (1.2 g (66% yield) of the product having a melting point of 217 12185X (with decomposition). [04,, 28.5 (c. l in water). The thus obtained product gave a positive ferric chloride reaction (red-purple color) and was identified as cyclo-dLN -acetyl-l l' hydroxy-L-onithyl (rhodotorulic acid) by its N.M.R. and IR. spectra shown in Table l below.

Analysis: Calcd. for H- N 0 yclo di N 5 l1cn7ylo\ \-D-ornithyl Dihydrobromide Example (1 IA). Cyclo-diN acetyLN -benzyl0xy-L-0rnithyl (178 g of cyclo di N dwemyloxyl.-ornithyl prepared as in Example 4 (B) was mixed with 6.5 ml of pyridine and (1.5 ml ol acetic anhydride. The resulting mixture was stirred for Ill hours at room temperature and then evaporated in vacuo. The thus obtained resi due as combined with water and the mixture was evaporated 4 this treatment being repeated twice) to decompose any remaining acetic anhydride present in the mi\ture in a small amount. The resulting residue Was taken into N) ml ot ethyl acetate. and the solution was washed repeatedly with water. The organic layer was dried over sodium sullatc and evaporated in vacuo to dryness. Retrystalli/ation ot' the thus obtained residue lroni ethanol tllttfl \icldcd cyclo-di-1' l' -acetyl-N hen/ loxyl oinitlryl melting point ol UT L)lj( liming a Yield: as; 1,, 264 lc. l in ethanol). Analysis. (.iled for C H NLL;

lound N.

(ill

C, 48.83; H, 7.03; N. I627 Found: C. 48.81; H. 7.07; N. 16.18.

Table l N.M.R. and IR. spectra of product N.M.R. Hs tF CCOOHI 7.9 it Side-chain ethylene 7.5 tr Acctyl methyl fit) 4 Side-chain methylene adjacent to nitrogen 55 2 Ring ucarbon l.fi 2 Ring amide [.R. lcm") 3l8tl l4llt) I85 lllll) 2860 l I51) 169(1 (amide l) Wit) lShtl (hydroxamic acid carbonyl) t 2l l-li tl 7W2 l-ihll ']h\) 1445 Example 7 Cyclo-cli-N 5 -acetyl-N 5 -hydroxy-D-ornithyl Following the procedure described in Example 6 (B) under the same conditions. but using cyclo-di-N 5 aeetyl-N -hen2yloxy-D-ornithyl in place of cyclo-di- N -acetylN ben7.yloxy-L-ornithyl. there was ob tained cyclo-di-N -acetyl-l l -hydroxy-D ornithyl having a melting point of 2l62l85.

Yield: 38.8%. [04],, +281) (C. do in water).

Analysis: Calcd. for C H N O C. 48.83: H. 7.03; N. I627 Found: (T. 48.72; H. (1.88: N. ltxtlS.

What we claim is:

I. A process for the production of cyclo-di-N alkanoyLN -h \droxy-ornith \l having the formula:

I-I H COR N o :N /\/\E/ N/ RCO 0 OH herein R represents a lower alkyl group having l to 4 carbon atoms. inclusive. which comprises the step of catalytically reducing an alkanoyl compound ot the for (VII) herein R is as defined above by hydrogenation in the 4 carbon atoms. lnClllhlYL. which comprises the steps of 'csence of palladiun'i-on-carhon at room temperature treating a compound of the formula N o :N/\/1 ZA N/O-CI-I2 O CHZO O O E so OCH 2. A process for the production of cyclo-di-N in a solvent system consisting ot hydrogen bromide canoyl-N' -hydroxy-ornithyl having the formula acetic acid and phenol wherein 30 to 40 percent by L10 1;! weight hydrogen bromide is in solution in the acetic N O C R acid and phenol is present in (Ll-(L3 parts by weight/l N part by weight acetic acid at l8 C to 25 C for at least .40

0 1?] VV OH thirty hours to produce a compound of the formula H (V111) (VI).

\erein R represents a lower alkyl group having I to then acylating the compound of the formula (V!) with :arhon atoms, inclusive. which comprises the steps of m an acid anhydride in quinoline or pyridine to produce ylating a compound having the formula a compound of the formula (VII) 'CHz -N r o I H N-O-CH N AM O Z- N OCH 0 I coa (VII) ith an acid anhydride in quinoline or pyridine to pro- 45 and then catalytically reducing the compound of the tee a compound of the formula formula (Vll) by hydrogenation in the presence of d then catalytically reducing the compound of the 55 palladium-on-carhon at room temperature.

'mula (Vll) by hydrogenation in the presence of 4. The process of claim 1 wherein R is methyl lladiumon-carhon at room temperature. 5. The process of claim 2 wherein R is methyl. 3. A process for the production of cyclo-di-N 6. The process of claim 3 wherein R is methyl. :anoyl-N' -hydroxy-ornithyl having the formula '7. Cyclo-di-N -tosyl-N -hcnzyloxy-ornithyl of H to t N (30R 0 N\ OH vm) terein R represents a lower alkyl group having I to the formula 

1. A PROCESS FOR THE PRODUCTION OF CYCLO-DI-N -ALKANOYLN -HYDROXY-ORNITHYL HAVING THE FORMULA:
 2. A process for the production of cyclo-di-N -alkanoyl-N -hydroxy-ornithyl having the formula
 3. A process for the production of cyclo-di-N -alkanoyl-N -hydroxy-ornithyl having the formula
 4. The process of claim 1 wherein R is methyl.
 5. The process of claim 2 wherein R is methyl.
 6. The process of claim 3 wherein R is methyl.
 7. Cyclo-di-N -tosyl-N -benzyloxy-ornithyl of the formula 